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Controlled antibody release from degradable thermoresponsive hydrogels cross-linked by Diels-Alder chemistry Manuel Gregoritza, Viktoria Messmann, Kathrin Abstiens, Ferdinand P. Brandl, and Achim M. Göpferich Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00587 • Publication Date (Web): 22 Jun 2017 Downloaded from http://pubs.acs.org on June 24, 2017

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Biomacromolecules

Controlled antibody release from degradable thermoresponsive hydrogels cross-linked by Diels-Alder chemistry

Manuel Gregoritza, Viktoria Messmann, Kathrin Abstiens, Ferdinand P. Brandl, and Achim M. Goepferich*

Department of Pharmaceutical Technology, Faculty of Chemistry and Pharmacy, University of Regensburg, 93040 Regensburg, Germany

Submitted to: Biomacromolecules

* Corresponding author: Phone: +49 941 943-4843; fax: +49 941 943-4807. E-mail address: [email protected] (Achim M. Goepferich) 1 Environment ACS Paragon Plus

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Graphical Abstract

Keywords hydrogel, dual gelation, Diels-Alder reaction, thermal gelation, controlled release, antibody delivery

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Abstract Amine-modified four- and eight-armed poloxamines were prepared and subsequently functionalized with maleimide or furyl groups. Aqueous solutions of these polymers exhibited an immediate gelation at a temperature above 37 °C. Concomitantly, Diels-Alder reactions 5

gradually cross-linked and cured the gels. Different ratios between four- and eight-armed macromonomers were used to tune hydrogel stability and mechanical properties. In this way, hydrogel stability could be precisely controlled in the range of 14 to 329 days. Controlled release of the model antibody bevacizumab was achieved over a period of 7, 21 and 115 days. Release profiles were tri-phasic with a low burst; approximately 87% of the released antibody

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was intact and displayed functional binding. The hydrogels presented in this study are degradable, non-toxic, rapidly gelling, stable and provide controlled antibody release. They can be tailored to match the demands of various applications and present an attractive platform for antibody delivery.

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(146/150 words)

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Introduction The Diels-Alder (DA) reaction is a highly attractive reaction for the design of in situ crosslinking hydrogels. DA reactions of maleimide and furan are particularly promising as they are efficient, require no catalysts, form no toxic side products, and can be carried out in water at 5

ambient temperatures.1 Recently, the DA reaction between maleimide and furan has been introduced as a cross-linking mechanism for the preparation of degradable poly(ethylene glycol) (PEG) based hydrogels.2 Overall, these hydrogels have outstanding potential for antibody delivery and controlled release. However, they are associated with relatively long gel times of 14 minutes or more due to the slow reaction kinetics of the DA reaction. As this

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is regarded a major drawback for future applications as injectable depot system, attempts have been made to accelerate gel formation, e.g., by utilizing hydrophobic association3 or by introducing additional branches.4 Although both approaches led to significant decreases in gel times, instantaneous gel formation could still not be achieved. As an alternative approach to design DA hydrogels that gel more quickly, the DA reaction

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could be combined with a second gelation mechanism that provides immediate gel formation upon injection. This could be achieved by using materials that respond to external stimuli, such as temperature, pH or ionic strength.5-8. For example, in situ forming hydrogels can be prepared using thermoresponsive block copolymers, e.g., poly(N-isopropylacrylamide), poloxamer (Pluronic®), and poloxamine (Tetronic®).9 These polymers exhibit a sudden

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reduction in solubility above a certain temperature, leading to rapid gel formation. This respective temperature is referred to as lower critical solution temperature (LCST). Therefore, the goal of this study was to develop thermoresponsive materials which can additionally undergo DA cross-linking. In this way, immediate gelation could be achieved, while at the same time, a major drawback of exclusively physically cross-linked gels can be eliminated:

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low resistance against dissolution and, thus, limited stability after administration.10-12


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For example, in a recent approach by Boere et al. thermal gelation was combined with native chemical ligation to obtain degradable hydrogels with controlled mechanical properties.13 However, until now, most attempts to combine thermal gelation and covalent cross-linking have involved Michael-type additions between acrylate and thiol groups. For example, Cellesi 5

et al. designed materials for cell encapsulation,14-16 Cho et al. developed tissue sealants17 and Censi et al. prepared a controlled release system for bradykinin.18 Although Michael-type additions between thiol and acrylate groups are suitable for dual gelation and in situ crosslinking, there are some disadvantages associated with the reaction that render it unsuitable for antibody delivery. For example, thiol groups on polymer chains can form dimers or alter the

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protein structure through thiol-disulfide exchange.19 Furthermore, thiol-Michael additions are often conducted in a basic environment to accelerate the reaction.20 However, high pH could be detrimental for antibody stability for example as many antibodies are formulated in acidic buffers, e.g., adalimumab (pH 5.2), ranibizumab (pH 5.5), trastuzumab (pH 6.0), and bevacizumab (pH 6.2).21

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In this study, degradable hydrogels for controlled antibody delivery were prepared based on a dual approach involving both thermal gelation and the DA reaction. After rapid gel formation at body temperature, DA reactions gradually cured and stabilized the hydrogel. In this way, in situ forming yet highly stable hydrogels could be obtained. To prepare four-armed macromonomers, Poloxamine, a tetrafunctional thermoresponsive polymer, was end-

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functionalized

with

maleimide

(4armPoloxamine-maleimide)

or

furyl

groups

(4armPoloxamine-furan). To prepare eight-armed macromonomers, branched poloxamines were

first

synthesized

and

then

end-functionalized

(8armPoloxamine-maleimide,

8armPoloxamine-furan). DA-Poloxamine hydrogels were prepared by dissolving the macromonomers in water using a polymer concentration of 30 wt.%. Different hydrogel types 25

were prepared using various ratios of 8armPoloxamine to 4armPoloxamine. In the first part of the study, rheological experiments were carried out to characterize gel formation and 5 Environment ACS Paragon Plus

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mechanical properties. In the next part, hydrogel stability was analyzed and cytotoxicity was assessed. Finally, hydrogels were loaded with the model antibody bevacizumab (Avastin®) to analyze the in vitro release. Bevacizumab was selected as intravitreal injections of this antibody are currently used to treat neovascular age-related macular degeneration and the 5

treatment might benefit from controlled long-term release. Finally, antibody binding ability after release was confirmed to exclude a negative influence of gel formation on antibody stability.

Materials and methods 10

Materials Boc-6-aminohexanoic acid and Fmoc-Lys(Boc)-OH were purchased from Bachem (Weil am Rhein, Germany). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from PanReac AppliChem (Darmstadt, Germany). Poloxamine (Tetronic® 1107) was provided by BASF SE (Ludwigshafen am Rhein, Germany). Deuterated chloroform (CDCl3),

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N,N'-dicyclohexylcarbodiimide (DCC), Eagle's minimum essential medium (EMEM), fetal calf serum (FCS), 3-(2-furyl)propanoic acid, N-hydroxysuccinimide (NHS), and Nmethoxycarbonylmaleimide were received from Sigma-Aldrich (Taufkirchen, Germany). Mouse fibroblast L-929 cells were a kind gift from the group of Prof. Armin Buschauer (University of Regensburg). Bevacizumab (Avastin®, 25 mg mL-1, Roche Ltd, Basel,

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Switzerland) was kindly provided by the hospital pharmacy of the University of Regensburg (Germany). All other chemicals were obtained from Merck KGaA (Darmstadt, Germany). Purified water was freshly prepared using a Milli-Q water purification system (Millipore, Schwalbach, Germany).

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1H-NMR spectroscopy 1H-NMR spectra were recorded in CDCl at 295 K using a Bruker Avance 300 spectrometer 3 (Bruker BioSpin GmbH, Rheinstetten, Germany).

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Synthesis of four-armed macromonomers The terminal poloxamine hydroxyl groups were first converted to amino groups,22 and then end-functionalized with maleimide and furyl moieties as previously described for poly(ethylene glycol) (PEG) based macromonomers.2,22 In brief, poloxamine (Tetronic® 1107) was converted to 4armPoloxamine-NH2 by means of a Mitsunobu reaction followed by

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hydrazinolysis. 4armPoloxamine-NH2 was then reacted with N-methoxycarbonylmaleimide to yield 4armPoloxamine-maleimide. The reaction of 4armPoloxamine-NH2 with 3-(2furyl)propanoic acid, DCC, and NHS led to 4armPoloxamine-furan. The degree of end-group functionalization among the batches was 76 ± 8% for 4armPoloxamine-maleimide and 81 ± 1% for 4armPoloxamine-furan as determined by 1H-NMR.

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Synthesis of eight-armed macromonomers Eight-armed poloxamines were synthesized through branching as previously described for PEG-based macromonomers.4 Briefly, 4armPoloxamine-NH2 was first reacted with FmocLys(Boc)-OH, DCC and NHS. The Fmoc-groups were removed and Boc-6-aminohexanoic 20

acid was reacted with the free amino-group via DCC/NHS chemistry. All Boc-groups were then removed and the free amino-groups were functionalized with furyl (8armPoloxaminefuran) or maleimide moieties (8armPoloxamine-maleimide) as described above. The degree of end-group functionalization among the batches was 64 ± 6% for 8armPoloxaminemaleimide and 82 ± 4% for 8armPoloxamine-furan as determined by 1H-NMR.

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O

O

N O

O X

y

N

O X

y N y

y

N

y

O

O N

O N

O

O

y

N O y

N H

O

O X

O

X

O

X

O

O

O

O y

X

O

H N O

O

O

O X

O

H N

O

N

O

O

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O

O

O

N H

X

O

4armPoloxamine-furan

4armPoloxamine-maleimide O O

O

O

O

N O

O

HN

N

NH

O

O

O

NH

H N

N O O

O X

O

y O

O

O

X

N O

O

NH

N H

O X

O N y

O

HN

H N O

O y

N H

O

O

O N H

O

O

H N O

O

O

O X

O

O

NH

N H

O X

O y N y

O y

O

O X

N H

HN

O N H

O

y

N

O

HN

H N

O

O

H N

N HN

NH

X

O O X

O

O O

O

y

N

O N

O O

H N O

O

O N O

NH

O O

O O

8armPoloxamine-maleimide

5

HN

O

N

O

8armPoloxamine-furan

Scheme 1: Four- and eight-armed poloxamines were end-functionalized with maleimide or furyl groups. Macromonomers were synthesized from Tetronic® 1107; the x (poly(ethylene glycol, PEG) to y (poly(propylene glycol, PPG) ratio was 70:30. Branching was achieved by introducing lysine and 6-aminohexanoic acid residues.

Gel formation and rheological properties 10

For hydrogel preparation, the respective polymers were dissolved in water using a total polymer concentration of 30 wt.%. Various ratios between 8armPoloxamine and 4armPoloxamine were used to prepare hydrogels with different properties. For example, in order to prepare 1000 mg of a hydrogel composed of 20 wt.% 8armPoloxamine and 10 wt.% 4armPoloxamine, i.e., a 20%/10% hydrogel, a total amount of 300 mg polymer was dissolved

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in 700 mg water. To be more precise, for this type of hydrogel 100 mg 8armPoloxaminemaleimide, 100 mg 8armPoloxamine-furan, 50 mg 4armPoloxamine-maleimide, and 50 mg 4armPoloxamine-furan were used. Immediate gel formation could be induced by increasing the temperature to 37 °C. Rheological characterization was performed on a TA instruments AR 2000 rheometer (TA

20

Instruments, Eschborn, Germany). Oscillatory shear experiments were carried out using a 40 mm parallel plate geometry with a 500 µm gap size at a constant frequency of 0.5 Hz. Polymer solutions were prepared as described above; a sample volume of 750 µL was used. 8 Environment ACS Paragon Plus

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Absolute values of the complex shear modulus (|G*|) and phase angles (δ) were recorded as a function of time. In order to characterize the thermoresponsive behavior of the polymer solutions, heating (20 °C to 37 °C) and cooling (37 °C to 20 °C) steps were included in the procedure. Water evaporation was minimized by using a solvent trap and by pipetting silicone 5

oil around the gap between the plates. However, in order to minimize the influence of drying on the obtained |G*| values, rheological experiments were limited to 100 minutes.

Swelling and degradation Swelling and degradation of DA-Poloxamine hydrogels was studied in 50 mM phosphate 10

buffer, pH 7.4 at 37 °C as previously described.4 Different ratios between 8armPoloxamine and 4armPoloxamine were used as described above; a polymer concentration of 30 wt.% was used for all hydrogels.

Hydrolytic stability of maleimides 15

The hydrolytic stability of 4armPoloxamine-maleimide and 8armPoloxamine-maleimide was investigated in phosphate buffer, pH 7.4 at 37 °C as previously described.3

Cytotoxicity In order to assess the cytotoxicity of DA-Poloxamine hydrogels a metabolic assay using the 20

reagent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was performed according to ISO 10993-5:2009 (Biological evaluation of medical devices, part 5: Tests for in vitro cytotoxicity). In brief, 300 µL of 0%/30%, 15%/15%, and 30%/0% hydrogels were prepared, cast into a 24-microtiter plate and allowed to cross-link overnight in cell culture conditions (37 °C, 5% CO2). On the next day, extracts were prepared by adding 2.0 mL of

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EMEM supplemented with 10% FCS to the hydrogels which were then incubated for 24 hours. Mouse fibroblast L-929 cells were seeded in 96-microtiter plates at a density of 10,000 9 Environment ACS Paragon Plus

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cells per well and allowed to adhere overnight. 100 µL of extract was added to each well. 0.1% SDS served as a negative control and pure medium as a positive control (n = 12). Cells were incubated with the test media for 24 hours after which the medium was removed and 200 µL of serum-containing medium with 1.5 mM MTT was added to each well. After 5 5

hours of incubation the MTT solution was removed and 200 µL PBS containing 10% SDS was added to each well. After 16 hours of incubation, absorbance at 570 and 690 nm was determined for each well using a FluoStar Omega fluorescence microplate reader (BMG Labtech, Ortenberg, Germany). The difference in absorbance at 570 nm and 690 nm was used to calculate the viability of cells. The viability of the treated cells was normalized to the value

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for the positive control, i.e., medium alone. Antibody release and analytics Release experiments were carried out as previously described.3 However, in this study the bevacizumab loading was 6.25 mg mL-1 and the polymer concentration used was 30 wt.%. Due to the high stability of some hydrogels, release experiments were only carried out using

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0%/30%, 5%/25%, and 10%/20% hydrogels. In order to investigate a potential negative influence of hydrogel preparation and cross-linking reactions on the stability of bevacizumab, the antibody was analyzed after release. Functional binding to vascular endothelial growth factor (VEGF) was investigated using a Shikari® Q-beva enzyme-linked immunosorbent assay (ELISA) kit (Matriks Biotek, Ankara, Turkey). The amount of binding antibody was

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compared to a solution containing the same concentration of fresh bevacizumab.

Statistical analysis All results are presented as mean ± standard deviation, based on the data obtained from at least n = 3 samples. Statistical significance was determined by means of one-way ANOVA,


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followed by Tukey’s post-hoc test using GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla, CA, USA). Differences were considered statistically significant at p < 0.05.

Results and discussion 5

Hypothesis and proof of concept The goal of this study was to develop hydrogels that combine thermal gelation and DA chemistry in a dual gelation process.

This combination has several advantages for the

delivery of therapeutic antibodies. Firstly, precursor solutions can easily be prepared by 10

dissolving the polymer components in water or buffer together with the antibody to be encapsulated. Secondly, due to the slow reaction kinetics of the DA reaction, the fluidity of the polymer solution will remain unaffected at room temperature for a time span long enough to allow convenient handling and application. Thirdly, the thermoresponsive behavior will lead to immediate gel formation at a site of injection. In that way, high amounts of antibody

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can be "encapsulated" in situ. Finally, in contrast to hydrogels that rely exclusively on thermal gelation, these hydrogels are expected to be significantly more stable concerning degradation as a result of covalent cross-linking. In a proof of concept experiment, the rheological properties of modified 4armPoloxamines (“DA-Poloxamine”) were investigated and compared to unmodified commercially available

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Tetronic® 1107 (“Poloxamine”). For this purpose, 30 wt.% polymer was dissolved in water and analyzed using a three-step temperature program (Fig. 1). Absolute values of the complex shear modulus (|G*|) and phase angles were recorded over time to monitor gel formation and stiffness. At 20 °C, both Poloxamine and DA-Poloxamine were in the liquid state which was confirmed by |G*| values of 0.7 ± 0.2 Pa for Poloxamine and 0.8 ± 0.4 Pa for DA-Poloxamine.

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The phase angles (δ) were 90.1 ± 0.2° (Poloxamine) and 88.5 ± 2.4° (DA-Poloxamine), and the loss modulus (G'') was larger than the storage modulus (G') indicating mainly viscous 11 Environment ACS Paragon Plus

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behavior. Upon increasing the temperature to 37 °C (+∆T), the |G*| values for both sample types immediately increased confirming their thermoresponsive properties. Directly after heating, the |G*| values were 48.6 ± 6.9 kPa for Poloxamine and 36.2 ± 9.8 kPa for DAPoloxamine; differences were not statistically significant (p < 0.05). At the same time, δ 5

strongly decreased for both samples indicating the formation of a viscoelastic gel (G' > G''). During the following incubation time at 37 °C, the stiffness of Poloxamine gels remained unchanged. In contrast, |G*| values for DA-Poloxamine gels increased to 67.0 ± 13.7 kPa. The increase in stiffness was attributed to covalent cross-linking reactions which occurred while already in the gel state. It could also be observed that the increase in |G*| was mainly

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governed by the strongly rising G'. The contribution of G'' was much weaker. In a third step, the temperature was lowered to 20 °C (-∆T). As expected, unmodified Poloxamine gels returned to the liquid state (|G*| = 0.7 ± 0.3 Pa, δ = 90.3 ± 0.2°, G' < G'') whereas DAPoloxamines remained in the gel state (|G*| = 15.3 ± 3.2 kPa, δ = 3.5 ± 0.3°, G' > G''). Taken together, the experiment confirmed the hypothesis that DA-Poloxamines exhibit thermal

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gelation and DA cross-linking in a dual gelation process.


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A

80,000

Poloxamine

B

90

80,000

75

60,000

DA-Poloxamine

90 75

60,000

45 +∆T

-∆T

|G*| (Pa)

40,000

60 40,000 +∆T

30

20,000

45 -∆T

0

0 0

50

100

150

200

15 0

0 0

Time (min)

10

30

20,000 15

5

δ (°)

|G*| (Pa)

60 δ (°)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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50

100

150

200

Time (min)

Figure 1: Rheological behavior of 30 wt.% Poloxamine (A) and DA-Poloxamine (B) solutions in the course of a three-step temperature program. Absolute values of the complex shear modulus (|G*|), represented as white squares, and phase angles (δ), represented as black circles, were recorded as a function of temperature and time at a 0.5 Hz oscillatory frequency. The initial temperature was set to 20 °C. After 10 min, the temperature was increased to 37 °C (+∆T) and kept constant for 180 min. In the third step, the temperature was again reduced to 20 °C (-∆T). Both polymer solutions displayed thermal gelation, however, only for DAPoloxamines stiffness increased during incubation and could be retained after cooling. For the sake of clarity, error bars are not shown in the diagram.

Gel formation and mechanical properties 15

In order to utilize DA-Poloxamines for antibody delivery, hydrogels with tunable properties would be desired. To this end, both four- and eight-armed macromonomers were employed for hydrogel preparation. In this way, hydrogels with different amounts of functional groups and, therefore, cross-linking densities could be obtained. In the following section, hydrogels composed of both 4armPoloxamines and 8armPoloxamines were analyzed for their thermal

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gelation properties and gel stiffness. In the nomenclature used to describe the compositions, the first number denotes the amount of 8armPoloxamine in the mixture, the second number refers to the amount of 4armPoloxamine. For example, in order to prepare 1000 mg of a 20%/10% hydrogel, 100 mg 8armPoloxamine-maleimide, 100 mg 8armPoloxamine-furan, 50 mg 4armPoloxamine-maleimide, and 50 mg 4armPoloxamine-furan were dissolved in 700 mg


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water. The total polymer concentration was 30 wt.% for all hydrogel types. This was identified as the minimum concentration to induce immediate thermal gelation. In the next experiment, the rheological behavior of hydrogels with different 8arm to 4arm ratios was analyzed (Fig. 2). |G*| was recorded at 20 °C for 10 min. Then, the temperature 5

was increased to 37 °C and kept constant for 90 min. Initially, all compositions were liquid and did not show significant stiffness; |G*| values ranged between 0.5 and 0.8 Pa. After 10 min at 20 °C the stiffness had slightly increased due to the beginning DA cross-linking, for example to 1.0 ± 0.6 for 0%/30% gels and to 1.7 ± 0.3 Pa for 20%/10% gels. Here, the slow reaction kinetic of the DA reaction is an advantage as faster covalent cross-linking could

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cause faster gel formation and might prevent administration, e.g., by clogging the needle. For example, hydrogels that rely on the relatively fast Michael-type addition reaction already exhibit gel formation after about 100 seconds at room temperature and neutral pH.23 Upon increasing the temperature to 37 °C, stable viscoelastic gels were formed immediately. |G*| values determined directly after increase in temperature are listed in Table 1. For all

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compositions, a strong increase in stiffness was observed. Interestingly, there was no significant difference between |G*| values directly after the increase in temperature (p < 0.05). Consequently, the strength of the thermally induced gel formation is governed by the backbone and is hardly affected by the number of functional end-groups on the macromonomer. The thermoresponsive properties were comparable for all compositions.

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150,000

20%/10% 15%/15%

|G*| (Pa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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125,000

10%/20% 15%/25%

100,000

10%/30%

75,000 50,000 25,000

+∆T

0 0

20

40

60

80

100

Time (min)

5

10

Figure 2: Initial thermal gelation and subsequent increase of stiffness for different 8armPoloxamine and 4armPoloxamine mixtures using an overall polymer concentration of 30 wt.%. Absolute values of the complex shear modulus (|G*|) were recorded over time at a 0.5 Hz oscillatory frequency. The initial temperature was set to 20 °C. After 10 min, the temperature was increased to 37 °C (+∆T) and then kept constant for 90 min. All polymer solutions were characterized by an immediate increase of stiffness at 37 °C. The further increase of stiffness was attributed to covalent cross-linking via Diels-Alder reaction and was stronger when more functional end-groups were present in the solution.

In contrast, the increase in stiffness after 90 minutes at 37 °C strongly depended on the 8arm/4arm-ratio used. For 0%/30% hydrogels, a |G*| increase of only 1.4-fold could be 15

observed. However, the |G*| increase was considerably more pronounced for hydrogels containing a higher concentration of branched macromonomers, e.g., 3.6-fold for 20%/10% hydrogels. This observation can be explained by the fact that hydrogel curing is caused by covalent DA cross-linking while already in the gel state. When more functional groups are present both the likelihood of covalent reactions and the maximum number of elastically

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active chains increases.


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Table 1: Absolute values of the complex shear modulus (|G*|) determined for hydrogels with different 8armPoloxamine to 4armPoloxamine ratios, measured directly after increase of temperature (0 min) and after incubation (90 min). Hydrogel composition

|G*| (0 min)

|G*| (90 min)

20%/10%

35.6 ± 10.3 kPa

126.7 ± 19.0 kPa

15%/15%

40.6 ± 1.3 kPa

114.0 ± 2.6 kPa

10%/20%

45.2 ± 3.7 kPa

94.0 ± 7.2 kPa

5%/25%

46.1 ± 3.9 kPa

83.0 ± 8.5 kPa

0%/20%

36.2 ± 9.8 kPa

52.1 ± 11.9 kPa

5

Hydrogel swelling and degradation In the previous experiments, it was demonstrated that DA-Poloxamines can be prepared using different amounts of four- and eight-armed macromonomers. For all compositions, immediate 10

gel formation at body temperature was observed. Hydrogel stiffness could be increased using a higher amount of 8armPoloxamine. Due to their rapid gelation and controllability, DAPoloxamine hydrogels present an attractive candidate as a "smart" material for drug delivery. However, in order to utilize these hydrogels for controlled antibody delivery, sufficient stability must be demonstrated. To this end, the swelling and degradation behavior of DA-

15

Poloxamine gel cylinders was analyzed in phosphate buffer, pH 7.4 at 37 °C (Fig. 3A).


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7 6

0%/30%

15%/15%

5%/25%

20%/10%

10%/20%

B

250

Onset of erosion (days)

A

Relative mass (mt/m0)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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5 4 3 2

y = 0.5524x 2 - 1.488x + 10.02 (R2 = 0.9988)

200

150

100

50

1 0

0 0

50

100 150 200 250 300 350

0

5

5

10

15

20

8armPoloxamine (wt.%)

Time (days)

Figure 3: Swelling and degradation of DA-Poloxamine hydrogels with different compositions in phosphate buffer, pH 7.4 at 37 °C (A). A higher number of functional endgroups correlated with an increase in stability and a lower degree of swelling. The onset of hydrogel erosion, i.e., beginning decrease of relative hydrogel mass, could be correlated with the concentration of 8armPoloxamine using a second-order polynomial (B).

Similar to hydrogel stiffness, the stability and swelling properties of DA-Poloxamine 10

hydrogel can be controlled by choosing the right composition. For example, hydrogels prepared based on four-armed macromonomers alone (0%/30%) completely dissolved after approximately 14 days. The maximum relative mass increase was 5.7 ± 1.0. In comparison, hydrogels containing the highest amount of branched macromonomers (10%/20%) completely dissolved after 329 days and reached a relative mass of only 2.9 ± 0.3.

15

In order to select the right composition for a certain application it would be highly desirable to be able to predict hydrogel stability. Therefore, the relationship between onset of hydrogel erosion and 8armPoloxamine content was investigated (Fig. 3B). The onset of erosion was defined as the time-point where the relative hydrogel mass started to decrease. Interestingly, the relation between 8armPoloxamine content and onset erosion can be described using a

20

second-order polynomial (R2=0.9988). This correlation can be used to predict the stability of a specific composition, or to tailor hydrogel stability for a given application. In order to understand why the relationship between 8armPoloxamine concentration and stability is not a 17 Environment ACS Paragon Plus

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linear but a quadratic one, it must be taken into account that there are two factors influencing DA-Poloxamine hydrogel stability.

O

O

H N N

O

R O

O

O

DA reaction R

N rDA reaction

R

O

NH O R

Ring-opening hydrolysis

OH-

O R

N H -O O

5

Scheme 2: Diels-Alder (DA) reactions of maleimide and furyl functionalized poloxamines (R) lead to covalent hydrogel cross-linking. Hydrogel degradation is mediated by retro-DielsAlder (rDA) reactions followed by the ring-opening hydrolysis of maleimide.

On the one hand, hydrogels containing a higher amount of branched macromonomers possess a higher number of functional end-groups and can, therefore, build up more elastically active 10

chains. Hydrogels with a higher cross-linking density exhibit a higher resistance to swelling and more cross-links have to be broken until hydrogels degrade. On the other hand, it has already been described by Kirchhof et al. that maleimide hydrolysis plays a pivotal role for DA-hydrogel stability (Scheme 2).24 As degradation occurs by retro-Diels-Alder (rDA) reaction and subsequent ring-opening hydrolysis of maleimide moieties, a higher hydrolytic

15

resistance is accompanied by an increase in hydrogel stability. In the course of synthesizing branched Poloxamines, lysine and 6-aminohexanoic acid residues were introduced between the polymer backbone and the maleimide moieties. According to literature, N-alkylation generally increases the hydrolytic stability of maleimides.25-28 This can be explained by the fact that electron donating N-substituents stabilize the C-N-C bond of maleimide and that


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enolization of the carbonyl group can be accelerated by N-alkylation.25 Consequently, nucleophilic attacks by hydroxide ions which lead to maleimide hydrolysis are hampered. This is in line with the experimental results obtained for 4armPoloxamine-maleimide and 8armPoloxamine-maleimide (Fig. 4). Maleimide hydrolysis was monitored for both 5

macromonomers in phosphate buffer, pH 7.4 at 37 °C. Assuming a pseudo-first order reaction, half-lives (t1/2) and rate constants (kobs) were calculated by fitting the integrated form of eqn (1) to the experimental data.

(1) 10

The results are listed in Table 2. On average, the half-life of 8armPoloxamine-maleimide was 2.8-fold higher than for 4armPoloxamine-maleimide. Interestingly, the half-life of 8armPoloxamine-maleimide (t1/2 = 874 min) was also considerably higher than for PEGbased macromonomers which were branched using the same chemistry (t1/2 = 412 min),4 or 15

3 macromonomers bearing an even longer C12 N-alkyl spacer (t1/2 = 585 min). Similarly,

4armPoloxamine-maleimide (t1/2 = 315 min) displayed a much longer half-life than PEG4 based maleimides, e.g., 8armPEG20k-maleimide (t1/2 = 177 min). These observations can be

explained by the more hydrophobic PEG-PPG polymer backbone used in this study. Taken together, the combined effects of retarded hydrolysis and increased cross-linking density 20

contribute

to

the

massive

stability

increase

observed

8armPoloxamine.


for

hydrogels

containing

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1.0

Rel. Absorbance (299 nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.8

0.6

0.4

0.2

4armPoloxamine-maleimide 8armPoloxamine-maleimide

0.0 0

200

400

600

800

1000

Time (min)

5

Figure 4: Absorbance of 4armPoloxamine-maleimide and 8armPoloxamine-maleimide in phosphate buffer, pH 7.4 at 37 °C. The absorbance decreased due to ring-opening hydrolysis of maleimide. Experimental data are indicated by box symbols; solid lines represent the best fits of eqn (1).

10

15

20

Table 2: Calculated rate constants (kobs) and half-lives (t1/2) for the ring-opening reaction of 4armPoloxamine-maleimide and 8armPoloxamine-maleimide in phosphate buffer, pH 7.4 at 37 °C. Macromonomer

kobs (min-1)

t1/2 (min)

R2

4armPoloxaminemaleimide

2.2010-3

315

0.9972

8armPoloxaminemaleimide

0.7910-3

874

0.9972


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Cytotoxicity Before application of DA-Poloxamine hydrogels in pre-clinical studies, toxicological safety should be demonstrated. PEG-PPG block copolymers like poloxamer (Pluronic) and poloxamine (Tetronic) are generally regarded as safe and are approved by regulatory 5

authorities such as the U.S. Food and Drug Administration.29-31 However, as the poloxamines were chemically modified in this study their toxicological potential should be re-assessed. In particular, leaching of side-products and reactants that could not be removed during purification might have toxic potential. In order to assess the cytotoxicity of DA-Poloxamine hydrogels, an MTT assay was performed. The test was

10

carried out using mouse fibroblast L-929 cells according to ISO 10993-5:2009. Extracts were prepared by incubating FCS containing medium with 0%/30%, 15%/15%, or 30%/0% hydrogels for 24 hours at 37 °C (Fig. 5). Cell viability after exposure to the extracts was referenced to pure medium (positive control); medium containing 0.1% (w/V) SDS served as a negative control. The relative cell viabilities were 93.9 ± 5.4 (0%/30%), 96.6 ± 5.7

15

(15%/15%), and 100.7 ± 5.8 (30%/0%). Therefore, incubation with extracts from DAPoloxamine hydrogels did not exhibit a strong negative effect on cell viability. However, hydrogels composed of 4armPoloxamine alone displayed a significantly higher cytotoxicity than hydrogels containing 8armPoloxamine (p < 0.05). This can be explained based on two effects: When macromonomers were not properly bound to the network or covalent cross-

20

links were broken, polymer can be eluted. As eight-armed macromonomers bear more functional end-groups they are bound more strongly to the network. Therefore, the likelihood for an elution of 4armPoloxamine during extraction is higher. This is reflected by a higher cellular toxicity. One potential source for the cytotoxicity of the extracted polymer is its surface-active properties that might interfere with the integrity of cell membranes. A second

25

source for cytotoxic effects could be the extraction of acidic Poloxamine-maleamic acid which could alter the pH of the cell culture medium. In summary, DA-Poloxamines displayed 21 Environment ACS Paragon Plus

Biomacromolecules

a very low cellular toxicity. However, in future in vivo investigations are necessary to confirm these results and to ensure biocompatibility.

120 100

Viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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*

80 60 40 20 0 0%/30% 15%/15% 30%/0%

5

SDS

Figure 5: L-929 cell viability after exposure to extracts from 0%/30%, 15%/15%, and 30%/0% DA-Poloxamine hydrogels was determined using an MTT assay. Cell culture medium containing 0.1% (w/V) SDS served as a negative control. A cell viability of < 70% (dotted line) was considered cytotoxic. * indicates statistically significant differences to all other groups.

10

Controlled antibody release In the final experiment, antibody release from DA-Poloxamine hydrogels was studied. To this end, three hydrogel compositions were loaded with the model antibody bevacizumab (Avastin) and the in vitro release was analyzed in phosphate buffer, pH 7.4 at 37 °C (Fig. 6). 15

As expected from swelling and degradation experiments, the release rate was strongly influenced by hydrogel composition. The least stable hydrogel (0%/30%) released the antibody after 7 days. When higher ratios of branched macromonomer was used for gel preparation, antibody release could be significantly delayed. Bevacizumab was released over the course of 21 days for 5%/25% hydrogels and 115 days for 10%/20% hydrogels. In all

20

three cases, more than 90% of the antibody that had been loaded could be recovered after release. Interestingly, the release profiles for the three hydrogel types all had a comparable shape, but on different time-scales. Initially, a notably low burst release of about 2 - 6% was 22 Environment ACS Paragon Plus

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observed. After a short lag phase, a rapid antibody release could be observed. The fast release can be explained by an increase of meshwork size resulting from hydrogel swelling. Consequently, antibodies were able to freely diffuse out of the network. The cumulative antibody release in the first phase was 67.9 ± 8.0 (0%/30%), 53.2 ± 4.9 (5%/25%), and 30.3 ± 5

3.9 (10%/20%). The amount of antibody released in this phase was higher for hydrogels with a lower number of covalent cross-links. In the next phase, antibody release was considerably slower. During this plateau phase, the majority of antibody was trapped within the polymer network. During this phase, single cross-links were broken over time due to rDA reaction and maleimide hydrolysis. Consequently, antibody release was strongly delayed. In case of the

10

most stable gel, this plateau phase spanned over 93 days. In the third phase, a rapid antibody release was observed. The constant breaking of cross-links led to a gradual increase of average meshwork size until a critical meshwork size had been exceeded. At this point, the previously entrapped antibody was rapidly released. A tri-phasic release profile could be beneficial for various applications. One example could be

15

in the treatment of ocular neovascularizations, in which anti-VEGF antibodies are directly administered into the vitreous body. As the ocular half-life of antibodies is 7-10 days32 sustained release systems could be used to provide sufficient antibody concentrations over weeks. However, as VEGF is a key player in maintaining cone photoreceptors and the choroidal vasculature, a constant blockage of VEGF signaling might lead to additional side-

20

effects. The intermittent release profile provided by DA-Poloxamine hydrogels might be an attractive alternative as VEGF-dependent structures can recover from the action of VEGFneutralizing antibodies during the plateau phase.33,34 Another potential application for DAPoloxamine hydrogels could be in the administration of vaccines. The intermittent release profile could be used to deliver antigens in a pulsed manner following a single

25

administration.35-37 Moreover, DA-Poloxamine hydrogels could most likely also be used for


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the controlled release of other biopharmaceuticals, e.g., immunoglobulin G with a size comparable to bevacizumab. Finally, as hydrogel preparation and cross-linking are known to potentially impair protein stability the binding ability of released bevacizumab was analyzed. To this end, bevacizumab 5

was incorporated into 0%/30% and 5%/25% hydrogels. The hydrogels were then allowed to fully degrade for 7 days and 30 days, respectively. Samples were taken and bevacizumab binding to VEGF was analyzed using ELISA. Fresh bevacizumab in the same concentration as the sample solution was used as a reference. As the focus was put on the influence of preparation and cross-linking, the antibody binding was not analyzed for 10%/20% hydrogels.

10

The long degradation time of more than 100 days would have influenced the results through unspecific effects, such as oxidation, deamidation, aggregation, and adsorption to the vial surface.38-40 For both hydrogel types, most of the released bevacizumab retained its binding properties (Table 3). The proportion of binding antibody was 87.0 ± 5.6% for 0%/30% hydrogels and 87.1 ± 1.7% for 5%/25% hydrogels. Consequently, preparation, thermal

15

gelation and cross-linking had only a minimal influence on antibody binding. Interestingly, when bevacizumab was incubated in phosphate buffer pH 7.4 for 30 days at 37 °C, the amount of binding bevacizumab was only 82.5 ± 2.7 %. When compared to bevacizumab released from 5%/25% hydrogels over 30 days, the amount of intact antibody was significantly lower (p < 0.05). Consequently, it can be concluded that DA-Poloxamine

20

hydrogels exhibit a protective effect against antibody degradation in vitro.


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A

120

Cumulative release (%)

100 80

60

40 20 0%/30% 0 0

2

4

6

8

10

Time (days)

B

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100 80

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80

60

40

20 10%/20% 0 0

20

40

60

80

100

120

140

Time (days)

Figure 6: Release of bevacizumab from 0%/30%, 5%/25%, and 10%/20% DA-Poloxamine hydrogels in phosphate buffer, pH 7.4 at 37 °C.


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To summarize, it could be demonstrated that DA-Poloxamine hydrogels are an outstanding material for the controlled release of therapeutic antibodies. Time-scales of release can be influenced by hydrogel composition. The general release profile was independent from hydrogel composition and is characterized by three phases and a low burst release. In all cases, 5

> 90% of the loaded bevacizumab was released and about 87% of the released antibody showed a binding comparable to fresh bevacizumab.

10

Table 3: Analytical investigation of bevacizumab released from DA-Poloxamine hydrogels. Bevacizumab binding to VEGF was quantified using ELISA; results were compared to a solution containing the same concentration of fresh bevacizumab. Hydrogel composition

Time-scale (days)

Binding antibody (%)

0%/30%

7

87.0 ± 5.6

5%/25%

30

87.1 ± 1.7

Conclusion 15

In this study, poloxamines were branched and end-functionalized with maleimide or furyl groups to yield four- and eight-armed macromonomers. At room temperature, aqueous solutions of these polymers were viscous fluids. Upon increasing the temperature to 37 °C, viscoelastic gels were immediately formed while subsequent DA reactions covalently crosslinked the gel. In this way, the many advantages of the DA reaction for hydrogel design could

20

be utilized while avoiding the drawback of slow gelation. In addition, the hydrogels presented in this work are degradable and non-toxic. Their mechanical properties can be precisely tailored to match the demands of various applications using appropriate compositions. Moreover, hydrogel stability could be adjusted between 14 days and 329 days; the onset of hydrogel erosion could be predicted using a second-order polynomial. Controlled in vitro

25

release of the model antibody bevacizumab was achieved over periods of 7, 30 and 115 days. 26 Environment ACS Paragon Plus

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In all cases, more than 90% of the loaded bevacizumab was released and 87% exhibited binding capabilities comparable to fresh bevacizumab.

Supporting Information 5

Supplementary spectroscopic data for the macromonomers used in this study.

Acknowledgments Financial support from the German Research Foundation (DFG), grant number BR 5132/1-2 is gratefully acknowledged. The authors thank Renate Liebl for technical assistance. 10

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